From sheep to mice to cells: Tools for the study of the sphingolipidoses

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann–Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.     

Exploring the interaction between small RNAs and R genes during Brachypodium response to Fusarium culmorum infection

The present study aims to investigate small RNA interactions with putative disease response genes in the model grass species Brachypodium distachyon. The fungal pathogen Fusarium culmorum (Fusarium herein) and phytohormone salicylic acid treatment were used to induce the disease response in Brachypodium. Initially, 121 different putative disease response genes were identified using bioinformatic and homology based approaches. Computational prediction was used to identify 33 candidate new miRNA coding sequences, of which 9 were verified by analysis of small RNA sequence libraries. Putative Brachypodium miRNA target sites were identified in the disease response genes, and a subset of which were screened for expression and possible miRNA interactions in 5 different Brachypodium lines infected with Fusarium. An NBS-LRR family gene, 1g34430, was polymorphic among the lines, forming two major genotypes, one of which has its miRNA target sites deleted, resulting in altered gene expression during infection. There were siRNAs putatively involved in regulation of this gene, indicating a role of small RNAs in the B. distachyon disease response.     

Structure and Functional Analysis of YcfD,a Novel 2-Oxoglutarate/Fe-Dependent Oxygenase Involved in Translational Regulation in Escherichia coli

The 2-oxoglutarate (2OG)/Fe^2 +-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain-containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently, other reactions have been identified, such as tRNA modification. The Escherichia coli gene, ycfD, was predicted to be a JmjC-domain-containing protein of unknown function based on primary sequence. Recently, YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double-stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed α-helical arm mediates dimerization of YcfD. We further have shown that 2OG binds to YcfD using isothermal titration calorimetry and identified key binding residues using mutagenesis that, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST (glutathione S-transferase)-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.     

beta-Endorphin: deletion of a single amino acid residue abolishes immunoreactivity but retains opiate potency.

Two synthetic analogs of camel β-endorphin, one with omission of Leu-14 and the other with omission of Asn-20, have been assayed for immunoreactivity by radioimmunoassay, opiate activity in the guinea pig ileum preparation and analgesic potency in mice. It was found that the omission analogs had no immunoreactivity, but retained significant biological activities. As far as we are aware, this is the first instance in which deletion of a single amino acid residue in a biologically active peptide abolished immunoreactivity.     

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.     

The Genesis of Ribosome Structure: How a Protein Generates RNA Structure in Real Time

Ribosomal subunit assembly is initiated by the binding of several primary binding proteins. Results from chemical modification studies show that 16S ribosomal RNA undergoes striking structural rearrangements when protein S17 is bound. For the first time, we are able to distinguish and order these structural rearrangements by using time-dependent chemical probing. Initially, protein S17 binds to a portion of helix 11, inducing a kink-turn in that helix that bends helix 7 toward the S17-helix 11 complex in a hairpin-like manner, allowing helix 7 to bind to protein S17. This structural change is rapidly stabilized by interactions at the distal and proximal ends of both RNA helices. Identifying the dynamic nature of interactions between RNA and proteins is not only essential in unraveling ribosome assembly, but also has more general application to all protein-RNA interactions.     

miR-421 promotes apoptosis and suppresses metastasis of osteosarcoma cells via targeting LTBP2

Increasing evidence has confirmed that microRNAs (miRs) are involved in tumor development and progression. A previous study reported that miR-421 could serve as a diagnostic marker in patients with osteosarcoma (OS). The present study explored the potential roles of miR-421 in the regulation of cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition of OS cells. Our results showed that miR-421 was upregulated in OS tissues and cell lines (MG63, U2OS, HOS, and Saos-2) compared with the corresponding adjacent tissues or human osteoblast cells hFOB1.19, while the latent transforming growth factor β-binding protein 2 (LTBP2) expression was reduced. In MG63 and U2OS cells, CCK8 assay displayed that cell proliferation was repressed by the miR-421 inhibitor, conversely increased by miR-421 mimics. Inhibition of miR-421 promoted cell apoptosis rate, caspase 3 activity, cleaved-caspase 3 (c-caspase 3) expression, and Bax/Bcl-2 ratio, restoration of miR-421 showed the opposite functions. Suppression of miR-421 blocked migration and invasion, whereas miR-421 overexpression promoted the migration and invasion of MG63 and U2OS cells. In addition, real-time polymerase chain reaction and Western blot analysis revealed that miR-421 negatively regulated E-cadherin expression, and positively regulated the expression of N-cadherin and vimentin. The luciferase reporter assay determined that miR-421 could target LTBP2-3′-UTR, and LTBP2 expression was regulated negatively by miR-421 both in mRNA and protein levels. Depletion of LTBP2 partly abolished the biological functions of miR-421 inhibitor in OS. In conclusion, miR-421 plays an oncogenic role in OS via targeting LTBP2, suggesting that miR-421 may be a potential therapeutic target against OS.